High-Affinity Binding of Chemokine Analogs that Display Ligand Bias at the HIV-1 Coreceptor CCR5.
Journal
Biophysical journal
ISSN: 1542-0086
Titre abrégé: Biophys J
Pays: United States
ID NLM: 0370626
Informations de publication
Date de publication:
03 09 2019
03 09 2019
Historique:
received:
15
03
2019
revised:
01
07
2019
accepted:
22
07
2019
pubmed:
20
8
2019
medline:
17
9
2020
entrez:
19
8
2019
Statut:
ppublish
Résumé
The chemokine receptor CCR5 is a drug target to prevent transmission of HIV/AIDS. We studied four analogs of the native chemokine regulated, on activation, normal T-cell-expressed, and secreted (RANTES) (CCL5) that have anti-HIV potencies of around 25 pM, which is more than four orders of magnitude higher than that of RANTES itself. It has been hypothesized that the ultrahigh potency of the analogs is due to their ability to bind populations of receptors not accessible to native chemokines. To test this hypothesis, we developed a homogeneous dual-color fluorescence cross-correlation spectroscopy assay for saturation- and competition-binding experiments. The fluorescence cross-correlation spectroscopy assay has the advantage that it does not rely on competition with radioactively labeled native chemokines used in conventional assays. We prepared site-specifically labeled fluorescent analogs using native chemical ligation of synthetic peptides, followed by bioorthogonal fluorescent labeling. We engineered a mammalian cell expression construct to provide fluorescently labeled CCR5, which was purified using a tandem immunoaffinity and size-exclusion chromatography approach to obtain monomeric fluorescent CCR5 in detergent solution. We found subnanomolar binding affinities for the two analogs 5P12-RANTES and 5P14-RANTES and about 20-fold reduced affinities for PSC-RANTES and 6P4-RANTES. Using homologous and heterologous competition experiments with unlabeled chemokine analogs, we conclude that the analogs all bind at the same binding site, whereas the native chemokines (RANTES and MIP-1α) fail to displace bound fluorescent analogs even at tens of micromolar concentrations. Our results can be rationalized with de novo structural models of the N-terminal tails of the synthetic chemokines that adopt a different binding mode as compared to the parent compound.
Identifiants
pubmed: 31421836
pii: S0006-3495(19)30648-4
doi: 10.1016/j.bpj.2019.07.043
pmc: PMC6731458
pii:
doi:
Substances chimiques
Chemokine CCL5
0
Chemokines
0
Ligands
0
Receptors, CCR5
0
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.
Langues
eng
Sous-ensembles de citation
IM
Pagination
903-919Subventions
Organisme : NIGMS NIH HHS
ID : T32 GM115327
Pays : United States
Informations de copyright
Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.
Références
Proc Natl Acad Sci U S A. 2000 May 9;97(10):5639-44
pubmed: 10779565
Science. 2004 Oct 15;306(5695):485-7
pubmed: 15486300
Proc Natl Acad Sci U S A. 2004 Nov 23;101(47):16460-5
pubmed: 15545608
Nat Rev Mol Cell Biol. 2004 Dec;5(12):998-1012
pubmed: 15573137
Proteomics. 2005 Apr;5(5):1199-203
pubmed: 15761952
Anal Biochem. 2005 Apr 15;339(2):271-81
pubmed: 15797568
J Biol Chem. 2006 May 5;281(18):12688-98
pubmed: 16476734
Nat Protoc. 2007;2(11):2842-56
pubmed: 18007619
J Immunol Methods. 2008 Feb 29;331(1-2):27-38
pubmed: 18054954
Mol Pharmacol. 2008 Mar;73(3):789-800
pubmed: 18096812
J Org Chem. 2008 Feb 1;73(3):983-91
pubmed: 18173281
J Mol Biol. 2008 Apr 4;377(4):1067-81
pubmed: 18313692
Protein Expr Purif. 2008 Oct;61(2):155-62
pubmed: 18588983
Proc Natl Acad Sci U S A. 2008 Nov 18;105(46):17706-11
pubmed: 19004761
J Microsc. 2008 Nov;232(2):343-52
pubmed: 19017233
J Biol Chem. 2009 Sep 25;284(39):26732-41
pubmed: 19542234
J Pharmacol Exp Ther. 2009 Oct;331(1):297-307
pubmed: 19584306
Mol Pharmacol. 2011 Mar;79(3):488-98
pubmed: 21088225
Biochemistry. 2011 Feb 1;50(4):502-11
pubmed: 21155586
Biophys J. 2011 Dec 21;101(12):2855-70
pubmed: 22208184
J Virol. 2012 Sep;86(18):10218-20
pubmed: 22787219
Retrovirology. 2013 Apr 20;10:43
pubmed: 23602046
Proc Natl Acad Sci U S A. 2013 Jun 4;110(23):9475-80
pubmed: 23696662
Science. 2013 Sep 20;341(6152):1387-90
pubmed: 24030490
Methods Cell Biol. 2013;117:267-303
pubmed: 24143983
J Biol Chem. 2014 Jul 4;289(27):19042-52
pubmed: 24855645
Sci Rep. 2014 Jun 26;4:5447
pubmed: 24965094
PLoS One. 2015 Apr 29;10(4):e0125396
pubmed: 25923671
Anal Biochem. 2016 Jun 1;502:24-35
pubmed: 26954998
Mol Pharmacol. 2016 Oct;90(4):483-95
pubmed: 27512119
J Am Chem Soc. 2016 Nov 30;138(47):15425-15433
pubmed: 27792324
Appl Transl Genom. 2013 May 26;2:3-16
pubmed: 27942440
Immunity. 2017 Jun 20;46(6):1005-1017.e5
pubmed: 28636951
Sci Signal. 2018 Oct 16;11(552):
pubmed: 30327411
Nature. 2019 Jan;565(7739):318-323
pubmed: 30542158
Nat Med. 2019 Jun;25(6):909-910
pubmed: 31160814
Biochemistry. 1984 Dec 18;23(26):6544-9
pubmed: 6529569
J Biol Chem. 1994 Mar 11;269(10):7224-30
pubmed: 7510283
Science. 1995 Dec 15;270(5243):1811-5
pubmed: 8525373
Nature. 1996 Jun 20;381(6584):667-73
pubmed: 8649512
Anal Biochem. 1995 Oct 10;231(1):269-71
pubmed: 8678314
Science. 1996 Sep 27;273(5283):1856-62
pubmed: 8791590
Nat Med. 1996 Nov;2(11):1240-3
pubmed: 8898752
Biophys J. 1997 Apr;72(4):1878-86
pubmed: 9083691
Science. 1997 Apr 11;276(5310):276-9
pubmed: 9092481
Chem Senses. 1997 Aug;22(4):467-76
pubmed: 9279469
Methods Enzymol. 1997;287:348-69
pubmed: 9330332